ABSTRACT (original parent award). Colon cancer is the second leading cause of cancer deaths among men and women combined. Therefore, alternative therapies are of critical need. In normal tissue, intestinal stem cells act to enable renewal of the entire epithelium every week. It is now known that the transformation of stem cells into colon cancer stem cells is a major inciting event that underlies carcinogenesis. Cancer stem cells are tumor-initiating cells, are refractory to therapy, and are linked to the very poor prognosis associated with late-stage colon cancer diagnosis. Better therapies may result if colon cancer stem cells could be eliminated. However, eradicating this population of cells is an extremely challenging problem, due to the intrinsic capacity of colon cancer stem cells to be long- lived, self-renewing, and highly proliferative. One proposed solution to this problem is to pharmacologically bias cancer stem cell fate toward a terminally differentiated cell fate. In other words, drive cancer stem cells to adopt a cell fate that does not proliferate, that has a short life expectancy, and that is sensitive to chemotherapy. Realization of this differentiation therapy requires that the identity of the cancer stem cell be known and that a candidate molecular target exists for a therapeutic entry point. To this end, the leucine-rich G protein coupled receptor-5 (Lgr5) has been found to act as a marker for colon cancer stem cells and is a tantalizing pharmacological target. Lgr5, together with its homologue Lgr4, act as modulators for the Wnt/catenin signaling pathway. Wnt/catenin signaling is a critical pathway that regulates stem cell homeostasis in the intestine. Mutations in this pathway result in excessive signaling and strongly bias stem cell behavior toward that of a long-lived and highly proliferative cancer stem cell. Lgr5 is therefore an attractive pharmacological target for counteracting this imbalance and restoring normal cell fate dynamics. However, the mechanisms of Lgr5-signaling and its roles in vivo are still vague and represent a major knowledge gap. Therefore, the objective of this proposal will be to determine how Lgr5-signaling controls stem and cancer stem cell fate. To accomplish this objective, I will test the central hypothesis that inhibition of Lgr5 internalization attenuates Wnt/catenin signaling and promotes cancer stem cell differentiation. Three specific aims have been proposed to test this hypothesis and will in (Aim 1) Clarify the mechanism of Lgr5-mediated Wnt/catenin signaling, (Aim 2) Elucidate Lgr5-dependent signaling mechanisms coordinating stem cell behavior, and (Aim 3) Identify small molecule interventions for driving cancer stem cell differentiation.